Ethanol production

ABSTRACT

This invention relates to ethanol production as a product of bacterial fermentation.

[0001] This invention relates to the production of ethanol as a productof bacterial fermentation. In particular, the invention relates toethanol production by thermophilic strains of Bacillus sp.

[0002] Many bacteria have the natural ability to metabolise simplesugars into a mixture of acidic and neutral fermentation products viathe process of glycolysis. Glycolysis is the series of enzymatic stepswhereby the six carbon glucose molecule is broken down, via multipleintermediates, into two molecules of the three carbon compound pyruvate.The glycolytic pathways of many bacteria produce pyruvate as a commonintermediate. Subsequent metabolism of pyruvate results in a netproduction of NADH and ATP as well as waste products commonly known asfermentation products. Under aerobic conditions, approximately 95% ofthe pyruvate produced from glycolysis is consumed in a number of shortmetabolic pathways which act to regenerate NAD⁺ via oxidativemetabolism, where NADH is typically oxidised by donating hydrogenequivalents via a series of steps to oxygen, thereby forming water, anobligate requirement for continued glycolysis and ATP production.

[0003] Under anaerobic conditions, most ATP is generated via glycolysis.Additional ATP can also be regenerated during the production of organicacids such as acetate. NAD⁺ is regenerated from NADH during thereduction of organic substrates such as pyruvate or acetyl CoA.Therefore, the fermentation products of glycolysis and pyruvatemetabolism include organic acids, such as lactate, formate and acetateas well as neutral products such as ethanol.

[0004] The majority of facultatively anaerobic bacteria do not producehigh yields of ethanol either under aerobic or anaerobic conditions.Most facultative anaerobes metabolism pyruvate aerobically via pyruvatedehydrogenase (PDH) and the tricarboxylic acid cycle (TCA). Underanaerobic conditions, the main energy pathway for the metabolism ofpyruvate is via pyruvate-formate-lyase (PFL) pathway to give formate andacetyl-CoA. Acetyl-CoA is then converted to acetate, viaphosphotransacetylase (PTA) and acetate kinase (AK) with theco-production of ATP, or reduced to ethanol via acetaldehydedehydrogenase (AcDU) and alcohol dehydrogenase (ADH). In order tomaintain a balance of reducing equivalents, excess NADH produced fromglycolysis is re-oxidised to NAD⁺ by lactate dehydrogenase (LDH) duringthe reduction of pyruvate to lactate. NADH can also be re-oxidised byAcDH and ADH during the reduction of acetyl-CoA to ethanol but this is aminor reaction in cells with a functional LDH. Theoretical yields ofethanol are therefore not achieved since most acetyl CoA is converted toacetate to regenerate ATP and excess NADH produced during glycolysis isoxidised by LDH.

[0005] Ethanologenic organisms, such as Zymomonoas mobilis and yeast,are capable of a second type of anaerobic fermentation commonly referredto as an alcoholic fermentation in which pyruvate is metabolised toacetaldehyde and CO₂ by pyruvate decarboxylase (PDC). Acetaldehyde isthen reduced to ethanol by ADH regenerating NAD⁺ Alcoholic fermentationresults in the metabolism of 1 molecule of glucose to two molecules ofethanol and two molecules of CO₂. The genes which encodes both of theseenzymes in Z. mobilis have been isolated, cloned and expressedrecombinantly in hosts capable of producing high yields of ethanol viathe synthetic route described above. For example; U.S. Pat. No.5,000,000 and Ingram et al (1997) Biotechnology and Bioengineerng 58,Nos. 2 and 3 have shown that the genes encoding both PDC (pdc) and ADH(adh) from Z. mobilis can be incorporated into a “pet” operon which canbe used to transform Escherichia coli strains resulting in theproduction of recombinant E. coli capable of co-expressing the Z.mobilis pdc and adh. This results in the production of a syntheticpathway re-directing E. coli central metabolism from pyruvate to ethanolduring growth under both aerobic and anaerobic conditions. Similarly,U.S. Pat. No. 5,554,520 discloses that pdc and adh from Z. mobilis canboth be integrated via the use of a pet operon to produce Gram negativerecombinant hosts, including Erwina, Klebsiella and Xanthomonas species,each of which expresses the heterologous genes of Z. mobilis resultingin high yield production of ethanol via a synthetic pathway frompyruvate to ethanol.

[0006] U.S. Pat. No. 5,482,846 discloses the simultaneous transformationof Gram positive Bacillus sp with heterologous genes which encode boththe PDC and ADH enzymes so that the transformed bacteria produce ethanolas a primary fermentation product. There is no suggestion that thebacteria may be transformed with the pdc gene alone.

[0007] A key improvement in the production of ethanol using biocatalystscan be achieved if operating temperatures are increased to levels atwhich the ethanol is conveniently removed in a vapourised form from thefermentation medium. However, at the temperatures envisioned,traditional mesophilic microorganisms, such as yeasts and Z. mobilis,are incapable of growth. This has led researchers to consider the use ofthermophilic, ethanologenic bacteria as a functional alternative totraditional mesophilic organisms. See EP-A-0370023.

[0008] The use of thermophilic bacteria for ethanol production offersmany advantages over traditional processes based upon mesophilic ethanolproducers. Such advantages include the ability to ferment a wide rangeof substrates, utilising both cellobiose and pentose sugars found withinthe dilute acid hydrolysate of lignocellulose, as well as the reductionof ethanol inhibition by continuous removal of ethanol from the reactionmedium using either a mild vacuum or gas sparging. In this way, themajority of the ethanol produced may be automatically removed in thevapour phase at temperatures above 50° C. allowing the production phaseto be fed with high sugar concentrations without exceeding the ethanoltolerance of the organism, thereby making the reaction more efficient.The use of thermophilic organisms also provides significant economicsavings over traditional process methods based upon lower ethanolseparation costs.

[0009] The use of facultative anaerobes also provides a number ofadvantages in allowing a mixed aerobic and anaerobic process. Thisfacilitates the use of by-products of the anaerobic phase to generatefurther catalytic biomass in the aerobic phase which can then bereturned to the anaerobic production phase.

[0010] The inventors have produced sporulation deficient variants of athermophilic, facultatively anaerobic, Gram-positive bacterium whichexhibit improved ethanol production-related characteristics.

[0011] This approach has a number of important advantages overconventional processes using both traditional and recombinant mesophilicbacteria, including simplification of the transformation process byusing only the pdc gene of Z. mobilis in strains that already produceethanol and have a ‘native’ adh gene. Expression of pdc has resulted ina significant increase in ethanol production by the recombinant organismand has unexpectedly improved the organism's growth characteristics.Recombinant microorganisms, which prior to transformation with the pdcgene were highly unstable and difficult to culture, show significantincreases in growth and survival rates both aerobically andanaerobically as well as an increase in the rate of ethanol productionnear to theoretical yields.

[0012] Accordingly, a first aspect of the invention relates to aGram-positive bacterium which has been transformed with a heterologouspdc gene, but which has solely a native alcohol dehydrogenase function.The gene may encode a functional equivalent of pyruvate decarboxylaseFunctional equivalents of pyruvate decarboxylase include expressionproducts of insertion and deletion mutants of natural pdc genesequences.

[0013] It is possible that organisms which carry out glycolysis or avariant thereof can be engineered, in accordance with the presentinvention, to convert as much as 67% of the carbon in a sugar moleculevia glycolysis and a synthetic metabolic pathway comprising enzymeswhich are encoded by heterologous pdc and native adh genes. The resultis an engineered organism which produces ethanol as its primaryfermentation product.

[0014] The Gram-positive bacterium is preferably a Bacillus. Thebacterium may be a thermophile. Where the Gram-positive bacterium is aBacillus it is preferably selected from B. stearothermophilus; B.calvodex; B. caldotenax; B. thermoglucosidasius, B. coagulans, B.licheniformis, B. thermodenitrificans, and B. caldolyticus.

[0015] A gene encoding lactate dehydrogenase may be inactivated in theGram-positive bacterium of the invention. For example, the lactatedehydrogenase gene may be inactivated by homologous recombination. Theheterologous pdc gene nay be from Zymomonas sp, preferably Z. mobilis ormay be from yeast e.g the S. cerevisae pdc 5 gene.

[0016] The heterologous gene may be incorporated into the chromosome ofthe bacterium. Alternatively, the bacterium may be transformed with aplasmid comprising the heterologous gene. Preferably the bacterium istransformed using plasmid is pFC1, more preferably with pFC1-PDC1. Theinvention includes Gram-positive bacteria, preferably a Bacillus spincluding the operon of the invention. The Bacillus sp may be selectedfrom B. stearothermophilus; B. calvodex; B. caldotenax, B.thermoglucosidasius, B. coagulans, B. licheniformis, B.thermodenitrificans, and B. caldolyticus. The operon may be incorporatedinto the genome of the Bacillus. Multiple copies of the PDC operon maybe incorporated into the genome.

[0017] In a preferred embodiment of the present invention theGram-positive bacteria has the heterologous gene operatively linked tothe lactate dehydrogenase promoter from Bacillus strain LN (NCIMBaccession number 41038) so that the heterologous gene is under thecontrol of the promoter. The sequence of the promoter region from strainLN is shown in FIG. 8.

[0018] The invention also provides strains LN (NCIMB accession number41038), LN-T (EB31, E32); TN (NCIMB accession number 41039); TN-P1;TN-P3; LN-S (J8) (NCIMB accession number 41040); LN-D (NCIMB accessionnumber 41041); LN-D11 and LN-P1.

[0019] According to another aspect of the invention, there is provided arecombinant, sporulation deficient, thermophilic Bacillus which grows atgreater than 50° C. The Bacillus is preferably not B. licheniformis.

[0020] A second aspect of the present invention relates to a method ofproducing ethanol using bacteria of the invention maintained undersuitable conditions.

[0021] The method may be operated at a temperature between 40-75° C.;preferably at a temperature of 52-65° C.; most preferably at atemperature of 60-65° C.

[0022] The present invention also relates to a method of producingL-lactic acid using strain LN.

[0023] The present invention also provides a nucleic acid moleculecomprising the lactate dehydrogenase promoter of strain LN (NCIMBaccession number 41038). The sequence of a nucleic acid moleculecomprising the lactate dehydrogenase promoter of strain LN is shown inFIG. 7. Preferably the nucleic acid molecule comprises a functionalfragment of the nucleic acid sequence shown in FIG. 7. A functionalfragment is defined as a fragment that function as a promoter andenables the expression of an operably linked gene.

[0024] The present invention also provides plasmid pFC1. The structureof this plasmid is shown schematically in FIG. 8

[0025] The present invention also provides plasmid pFC1-PDC1. Thestructure of this plasmid is shown schematically in FIG. 9.

[0026] The production of recombinant bacteria in accordance with theinvention will now be described, by way of example only, with referenceto the accompanying drawings, FIGS. 1 to 10 in which:

[0027]FIG. 1 is a schematic representation showing the production ofbacterial strains of the invention;

[0028]FIG. 2 illustrates the metabolic pathway whereby sugars aremetabolised to produce ethanol by bacterial strains of the invention;

[0029]FIG. 3 is a schematic representation illustrating the method ofLDH gene inactivation by single crossover recombination;

[0030]FIG. 4 is a schematic representation illustrating the method ofLDH gene inactivation by double crossover recombination;

[0031]FIG. 5 is a schematic representation illustrating the method ofLDH gene inactivation and heterologous gene expression by doublecrossover recombination;

[0032]FIG. 6 provides details about the PDC gene and promoter construct;

[0033]FIG. 7 provides sequence data about the LDH promoter from BacillusLN;

[0034]FIG. 8 is a schematic diagram of the replicative plasmid pFC1;

[0035]FIG. 9 is a schematic diagram of the PDC construct 2 cloned intothe pFC1 plasmid; and

[0036]FIG. 10 shows the construction of an artificial PDC operon.

[0037] The inventors initiated a strain development program to overcomeinherent strain limitations in respect of ethanol production, such asinstability and sporulation under adverse conditions. Physiologicalmanipulation and selection for strains with superior growthcharacteristics has been achieved in continuous culture, whereas a moretargeted genetic approach has been used to engineer strains with greaterstability and superior production characteristics.

[0038] In accordance with FIG. 1, all strains were ultimately derivedfrom a previously isolated Bacillus isolate PSII, a novel, thermophilic,Gram-positive, spore-forming, facultative anaerobe. PSII ferments a widerange of organic compounds from sugars, including hemicelluloses, toorganic acids such as acetate, formate and lactate and small amounts ofethanol at temperatures between 50° C. and 70° C.

[0039]Bacillus strains LLD-15, LLD-R, LLD-16 and T13 have been describedin EP-A-0370023 and by Amartey or al, (1999) Process Biochemistry 34 No.3 pp 289-294. Strain LLD-15 (NCIMB12428) arose during attempts to obtainmutants of Bacillus stearothermophilus strain NCA 1503 lacking L-LDHactivity by selecting for suicide substrate resistance (Payton andHartley (1984) Trends in Biotechnology, 2 No. 6). Strain LLD-15 wasassumed to be a variant of B. stearothermophilus NCA1503, but is, infact, derived from PSII. Strain LLD-R arises spontaneously andreproducibly from strain LLD-15 and is selected on plates or duringcontinuous cultures under which it grows more rapidly (i.e. at low pH inmedia containing sugars, acetate and formate). LLD-R produces L-lactateanaerobically and contains high levels of L-LDH, so is therefore,clearly a wild type revertant of the non-lactate-producing LLD-15lesion.

[0040]Bacillus strain T13 is an L-lactate deficient mutant of strainLLD-R. Isolation and characterisation of this mutant strain has beendescribed previously by, Javed. M. PhD Thesis, Imperial College, London.Thus, Bacillus strains LLD-15, LLD-16 and T13 are mutants of theoriginal isolate, PSII in which the ldh gene has been inactivated eithervia spontaneous mutation or by chemical mutagenesis and the majorfermentation product, unlike PSII, is ethanol. The lactate dehydrogenasegene mutation in LLD-15, LLD 16 and T13 results from the insertion of atransposon into the coding region of the lactate dehydrogenase generesulting in ldhT geno-type. All three strains are inherently unstablein high sugar concentrations (>2%) and revert back to lactate producingstrains. Strains T13 and LLD-15 revert to T13-R and LLD-R, respectively.Strains LLD-15, LLD-R, LLD-16 and T13 also tend to sporlate underadverse growth conditions such as changes in pH, temperature and mediumcompositions, and during periods of nutrient starvation. Sincesporulation often leads to culture washout in a continuous system, thesestrains are not suitable for large scale industrial use in which processparameters fluctuate.

EXAMPLE 1 Production of LN

[0041]Bacillus strain LN was produced from LLD-R as a spontaneoussporulation mutant which arose during nitrogen adaptation in continuousculture. Strain LN is more robust than the parental strains LLD-15 andLLD-R under vigorous industrial conditions such as low pH, high sugarconcentrations and in crude hydrolysate feed stocks and is more amenableto plasmid transformation. Strain LN is sporulation deficient (spo⁻) andis particularly suitable for the production of high purity L-lacticacid, producing up to 0.4 g of L-lactate/g of glucose at 65° C. Thisstrain is sensitive to kanamycin concentrations in excess of 5 μg/ml andan ideal host for genetic manipulation (ldh inactivation andbeterologous gene expression). Strain LN has been deposited under theterms of the Budapest Treaty under accession No. NCIMB 41038.

EXAMPLE 2 Production of Strain LN-T (E31/E32)

[0042] Strains E31 and E32 are spontaneous transposon mutants fromstrain LN. Both strains are lactate deficient and produce up to 0.5 g ofethanol/g of glucose at 65° C. They are non-sporulating and as such moreamenable to genetic manipulation than T13.

EXAMPLE 3 Production of Strain LN-S (J8)

[0043] Strain LN-S (J8) was produced by a single crossover homologousrecombination event between pUBUC-LDH a temperature sensitive,non-replicative plasmid harbouring an internal region of the ldh geneand the ldh gene on the chromosome (see FIG. 3). This resulted ininactivation of the ldh gene and a lactate negative phenotype. Thisstrain is also sporulation deficient and resistant to kanamycin. It isstable in relatively high sugar concentrations in continuous culture(with 10 g/L of residual sugar), it has good growth characteristics andproduces relatively high yields of ethanol. For example, in continuousculture at pH 7.0, 65° C., dilution rate of 0.1 h⁻¹, and 50 g/L glucosefeed, the cells produce up to 20 g/L of ethanol (i.e. 0.4 g of ethanol/gof glucose utilised) for 200 hours or more without any drop in ethanolyield

[0044] Strain LN-S (J8) has been deposited with the NCIMB under theterms of the Budapest Treaty under accession number NCIMB 41040.

EXAMPLE 4 Production of Strain LN-D

[0045] This strain was produced by a double crossover homologousrecombination event between a linear insertion cassette and the ldh genefrom strain LN (see FIG. 4). The insertion cassette (pUC-IC) is anon-replicating pUC18 plasmid harbouring a kranamycin resistance geneflanked by the ldh and lactase permease (1p) gene seequences.Recombination inactivated both the ldh and 1p genes resulting in alactate negative phenotype. This strain is also sporulation deficientand resistant to kanamycin. Strain LN-D can tolerate high sugarconcentrations (with up to 10 g/L of residual sugar), it has good growthcharacteristics and produces relatively high yields of ethanol. Forexample, in continuous culture at pH 7.0, 52° C., dilution rate of 0.1h⁻¹, and 50 g/L glucose feed, the cells produce Lip to 20 g/L of ethanol(i.e. 0.4 g of etbanol/g of glucose utilised) for 200 hours or morewithout any drop in ethanol yield or cell viability. Furthermore,kanamycin selection was not required to maintain the ldh geneinactivation.

[0046] Strain LN-D has been deposited with the NCIMB under the terms ofthe Budapest Treaty under accession number NCIMB 41041.

EXAMPLE 5 Production of Strain LN-D11

[0047] The resistance of strain LN-D to kanamycin was cured afterrepeated subculture to produce strain LN-D11. This strain is lactatenegative, sporulation deficient and sensitive to kanamycinconcentrations in excess of 5 μg/ml. Strain LN-D11 can tolerate highsugar concentrations (with up to 10 g/L of residual sugar), it has goodgrowth characteristics and produces relatively high yields of ethanol.For example, in continuous culture at pH 7.0, 52° C., dilution rate of0.1 h⁻¹, and 50 g/L glucose feed, the cells produce up to 20 g/L ofethanol (i.e. 0.4 g of ethanol/g of glucose utilised) for 200 hours ormore without any drop in ethanol yield or cell viability. This strain isan ideal host for heterologous gene expression.

EXAMPLE 6 Production of Strain LN-DP1

[0048] Strain LN-DP1 was produced from strain LN-D11 aftertransformation with the replicative plasmid pBST22-zym (also referred toas pZP1). The backbone of this vector is pBST22 (originally developed byLiao et al (1986) PNAS (USA) 83: 576-580) with the entire pdc gene fromZ. mobilis under the control of the ldh promoter sequence from B.stearothermophilus NCA 1503 Strain LN-DP1 is sporulation deficient;kanamycin resistant, lactate negative and contains the heterologous pdcgene from Z. mobilis. Strain LN-DP1 can utilise high sugarconcentrations, it has good growth characteristics and producesrelatively high yields of ethanol. For example, in continuous culture atpH 7.0, 60° C., dilution rate of 0.1 h⁻¹, and 50 g/L glucose feed, thecells produce up to 25 g/L ethanol (i.e. 0.5 g of ethanol/g of glucoseutilised) for 200 hours or more without any drop in ethanol yield.

EXAMPLE 7 Production of Strain TN

[0049] Strain TN arose spontaneously from T13 during nitrogen adaptationin continuous culture. This strain is more robust than the parentalstrain under vigorous industrial conditions (i.e. low pH, high sugarconcentrations, and in crude hydrolysate feed stocks) and is moreamenable to plasmid transformation, making it an ideal host for geneticengineering. Strain TN is sporulation deficient and lactate negative(the ldh gene has been inactivated by transposon mutagenesis into thecoding region of the ldh gene). Strain TN is a good ethanol producer ondilute sugar feeds. For example, in continuous culture at pH 70, 70° C.,dilution rate of 0.1 h⁻¹, and 20 g/L glucose, the cells produce up to 8g/L ethanol (i.e. 0.4 g of ethanol/g of glucose utilised) for 100 hoursor more without any drop in ethanol yield. This strain is sensitive tokanamycin concentrations in excess of 5 μg/ml and an ideal host forgenetic manipulation.

[0050] Strain TN has been deposited at the NCIMB under the terms of theBudapest Treaty under accession number NCIMB 41039.

EXAMPLE 8 Production of Strain TN-P1

[0051] Strain TN-P1 was produced from strain TN after transformationwith the replicative plasmid pBST22-zym. As previously described,pBST22-zym was produced by the incorporation of a pdc gene in vectorpBST22. The transformation efficiency of TN was 10-fold higher withpBST22-zym than pBST22 and the colony size of the transformants weresignificantly larger indicating that pdc expression conferred a growthadvantage to the cells.

[0052] TN-P1 is also more stable and a better ethanol producer than TN,especially in sugar concentrations greater than 20 g/L. For example, incontinuous culture controlled at pH 7.0, 52° C. with a dilution rate of0.1 h⁻¹, and a sugar feed containing 20-50 g/L, strain TN-P1 produced upto 0.5 g ethanol/g sugar utilised for 600 hours with no significant dropin yield or cell viability. In addition, in continuous culturecontrolled at pH 7.0, 52° C. with a dilution rate of 0.1 h⁻¹, and awheat crude hydrolysate feed, TN-P1 produced 0.4 g ethanol/g sugarutilised in for 400 hours with no significant drop in ethanol yield orculture viability.

[0053] Plasmid selection in strain TN-P1 was first maintained withkanamycin but the plasmid was found to be relatively stable withoutselection and no significant plasmid loss was detected after 300 hoursof continuous culture. The plasmid and PDC enzyme were found to berelatively stable at high temperatures. In continuous culture controlledat pH 7.0, with a dilution rate of 0.1 h⁻¹, and a glucose feed of 30g/L, there was no significant drop in ethanol yield or culture viabilitywhen the growth temperature was increased from 52 to 60°0) C.

EXAMPLE 9 Production of Strain TN-P3

[0054] Strain TN-P3 was produced from strain TN after transformationwith the replicative plasmid pFC1-PDC1. The backbone of this vector ispFC1 (FIG. 8) which was formed from a fusion of pAB124 (Bingham et al.,Gen. Microbiol., 114, 401-408, 1979) and pUC18. The pdc gene wasamplified from Z. mobilis chromosomal DNA by PCR using the followingprimers. The restriction sites BamHI and SacI (underlined) wereintroduced into the amplified gene. 5′-GAGCTCGCAATGAGTTATACTGTC-3′5′-GGATCCCTAGAGGAGCTTGTTA-3′

[0055] The ldh promoter was amplified by PCR from Bacillus LN using thefollowing primers. The restriction sites SacI and BamHI (underlined)were introduced into the amplified sequence.5′-GGATCCGGCAATCTGAAAGGAAG-3′ 5′-GAGCTCTCATCCTTTCCAAAA-3′

[0056] The ldh promoter sequence and pdc gene were digested with SacIand BamHI and then ligated together (FIG. 6). The construct was thendigested with BamHI and ligated into BamHI digested pFC1 to form plasmidpFC1-PDC1 (FIG. 9).

[0057] Strain TN-P3 is a good ethanol producer and produces yields inexcess of 0.45 g ethanol/g sugar at temperatures between 50 and 60° C.

[0058] Gene Inactivation

[0059] Singe-Crossover Recombination (SCO) (FIG. 3)

[0060] SCO or Campbell-type integration was used for directed ldh geneinactivation. An internal fragment (700 bp) of the target gene (ldh) wasfirst cloned into pUBUC to form pUBUC-LDH.

[0061] Plasmid pUBUC is a shuttle vector for DNA transfer betweenEscherichia coli and Bacillus strains LN and TN and was formed from thefusion of pUB110 and pUC18. This vector contains a selectable markerthat confers resistance to kanamycin and a Gram-positive andGram-negative replicon. The plasmid is temperature sensitive in Bacillusand cannot replicate above 54° C.

[0062] Plasmid PUBUC-LDH was first methylated (in vivo) and thentransformed into the host strain (LN) at the permissible temperature(50° C.) for plasmid propagation. The growth temperature was thenincreased to 65° C., preventing plasmid replication and integrants wereselected using kanamycin. Integration of the plasmid DNA into the ldhgene resulted in gene inactivation and a lactate negative phenotype.

[0063] Double-Crossover Recombination CDO) (FIGS. 4 & 5)

[0064] DCO or replacement recombination differs from SCO in that itresults in integration of only one copy of the target DNA and typically,a region of chromosomal DNA is replaced by another region, eitherforeign DNA or mutationally altered homologous DNA. The target DNA(kanamycin marker) was flanked on either side by mutationally alteredfragments of the ldh gene in plasmid pUC-IC (a non-replicative vectorbased on pUC18). The vector was first methylated (in vivo) and thenlinearised at a site outside the flanking region (this prevents SCO).The methylated, linearised plasmid DNA was then transformed into strainLN and integrants were selected using kanamycin.

[0065] This technique has also be used to inactivate ldh and integrate acopy of the pdc gene into the chromosome simultaneously (FIG. 5) and canbe applied to other genes of interest.

EXAMPLE 10 PDC Expression

[0066] In the expression plasmid pBST22-zym, the pdc gene from Z.mobilis is under the control of the ldh promoter sequence from B.stearothermophilus NCA1503 (FIG. 6). This plasmid was transformed intostrain TN to form TN-P1. Although PDC improved cell growth, pdcexpression and subsequent enzyme activity was relatively weak and therewas only a small increase in ethanol yield. In addition, PDC activity issensitive to temperature and rapidly declined at growth temperaturesgreater than 60° C.

[0067] Therefore, we increased expression of pdc by firstly replacingthe ldh promoter from B. stearothermophilus NCA 1503 with the ldhpromoter sequence from Bacillus sp. LN (FIG. 6). The pdc gene under thecontrol of ldh promoter from strain LN (construct 2) was subcloned intopFC1 to form plasmid pFC1-PDC1 (FIG. 9). Plasmid pFC1 is a shuttlevector that contains a Gram-positive and Gram-negative replicon andconfers resistance to ampicilin and tetracycline. This plasmid wastransformed into strain TN to form TN-P3.

[0068] Strain Performance

[0069] TN-P3 produces significantly more ethanol than the untransformedcontrol strain TN. Strain Ethanol Concentration TN 21.5 mM TN-P1 24.0 mMTN-P3 39.5 mM

[0070] The strains were cultured in a culture medium containing JSDsupplemented with 50 mM PIPES buffer and 2% glucose at 54° C. for 24hours. TNT-P1 and TN-P3 cultures were supplemented with kanamycin (12μg/ml) and tetracycline (10 μg/ml), respectively.

[0071] The thermostability of pdc can be improved by cloning analternative pdc gene, encoding a more thermostable PDC enzyme, fromSaccharomyces cerevisiae. This gene, referred to as pdc5 will also beunder the control of the ldh promoter from strain LN (see FIG. 9,construct 3). The construct will be cloned into pDC1 and the resultingplasmid pFC1-PDC5 will be transformed into strain TN.

[0072] Development of an Artificial PDC Operon

[0073] An artificial PDC operon can be constructed using interchangeablegene sequences from the ldh promoter, pdc genes from Z. mobilis and theyeast Saccharomyces cerevisiae, and adh from LN (see FIG. 10).

[0074] 1) pdc expression should be increased when the ldh promoter fromB. stearothermophilus NCA1503 is replaced by the ldh promoter from LNand will result in higher ethanol yields.

[0075] 2) the ethanol yields should be increased further if adh from LNis co-expressed with pdc from Z. mobilis in a PDC operon Ethanol yieldsshould be close to theoretical values of 0.5 g ethanol/g sugar.

[0076] 3) the thermostability of pdc may be increased above 64° C. ifthe Z. mobilis pdc gene is replaced by the pdc5 gene from S. cerevisiae.This will increase the growth temperature from 64 to70° C.

[0077] Integration of the PDC Operon into Strain LN-D11

[0078] By fusing the PDC operon to the insertion element sequence (firstidentified in the ldh gene from TN) several copies of the PDC operon canbe introduced into multiple sites on the chromosome increasing both thestability and gene dosage.

[0079] This expression strategy may also be used for other genes ofinterest.

1 5 1 24 DNA Z. mobilis 1 gagctcgcaa tgagttatac tgtc 24 2 22 DNA Z.mobilis 2 ggatccctag aggagcttgt ta 22 3 23 DNA Bacillus LN 3 ggatccggcaatctgaaagg aag 23 4 21 DNA Bacillus LN 4 gagctctcat cctttccaaa a 21 5301 DNA Bacillus LN 5 agggcaatct gaaaggaagg gaaaattcct ttcggattgtccttttagtt atttttatgg 60 ggagtgaata ttatataggc attacggaaa tgataatggcagagtttttt catttattag 120 actgcttgat gtaattggat gtgatgatac aaaaataatgttgtgtaaac aaaatgttaa 180 caaaaaagac aaatttcatt catagttgat acttcataaacattctcaaa taatccacaa 240 tatatcaatg tatgagcagt ttcacaaatt cattttttggaaaggatgac agacagcgat 300 g 301

1. A Gram-positive bacterium which has been transformed with aheterologous gene encoding pyruvate decarboxylase, wherein theheterologous gene expresses an active pyruvate decarboxylase, andwherein the bacterium has native alcohol dehydrogenase function.
 2. AGram-positive bacterium according to claim 1 wherein the bacterium is aBacillus sp.
 3. A Gram-positive bacterium according to claim 1 whereinthe bacterium is a thermophile.
 4. A Gram-positive bacterium accordingto claim 2 wherein the Bacillus is selected from B. stearothermophilus;B. calvodax; B. caldotenax, B. thermoglucosidasius, B. coagulans, B.licheniformis, B. thermodenitrificans, and B. caldolyticus.
 5. AGram-positive bacterium according to claim 1 wherein the gene encodinglactate dehydrogenase expression has been inactivated.
 6. AGram-positive bacterium according to claim 5 in which the lactatedehydrogenase gene has been inactivated by homologous recombination. 7.A Gram-positive bacterium according to claim 1 in which the heterologousgene is from Zymomonas sp or from Saccharomyces cerevisiae.
 8. AGram-positive bacterium according to claim 7 in which the heterologousgene is from Z. mobilis.
 9. A Gram-positive bacterium comprising anative adh gene and which has been transformed with a pdc 5 gene from S.cerevisiae.
 10. A Gram-positive bacterium according to claim 9 whereinthe heterologous gene is incorporated into the chromosome of thebacterium.
 11. A Gram-positive bacterium according to claim 1 in whichthe bacterium has been transformed with a plasmid comprising theheterologous gene.
 12. A Gram-positive bacterium comprising a native adhgene and which has been transformed with a plasmid comprising aheterologous gene encoding pyruvate decarboxylase, wherein the plasmidis pFC1.
 13. A Gram-positive bacterium comprising a native adh gene andwhich has been transformed with a heterologous gene encoding pyruvatedecarboxylase wherein the heterologous gene is operatively linked to thelactate dehydrogenase promoter from Bacillus strain LN (NCIMB accessionnumber 41038).
 14. Strains LN (NCIMB accession number 41038); LN-T (E31,E32); TN NCIMB accession number 41039); TN-P1; TN-P3; LN-S (J8) (NCIMBaccession number 41040); LN-D (NCIMB accession number 41041); LN-D11 andLN-DP1.
 15. The gram-positive bacterium of claim 9 wherein the bacteriumis a thermophile.
 16. The gram-positive bacterium of claim 12 whereinthe bacterium is a thermophile.
 17. The gram-positive bacterium of claim13 wherein the bacteria is a thermophile.
 18. The gram-positivebacterium of claim 9 further comprising an inactivated lactatedehydrogenase gene.
 19. The gram-positive bacterium of claim 12 furthercomprising inactivated lactate dehydrogenase gene.
 20. The gram-positivebacterium of claim 13 further comprising inactivated lactatedehydrogenase gene.